Cytochrome P-450 is the terminal oxidase of one of the monooxygenase systems found in the endoplasmic reticulum of many tissues. This monooxygenase catalyzes the oxidative metabolism of a wide variety of organic compounds including polycyclic aromatic hydrocarbons, fatty acids, steroids and drugs. Most of these reactions, like drug detoxification, are beneficial but some like the epoxidation of polycyclic aromatic hydrocarbons are not because these compounds are then carcinogenic. In no case is the metabolic control of this enzyme system understood. The objective of this Research Proposal is to study the mechanism and control of cytochrome P-450 catalyzed reactions. The enzyme source used will be the bacterium Pseudomonas putida because its cytochrome P-450 system is soluble and readily purified and has been previously shown to be an excellent model for the comparable mammalian membrane bound enzyme. The research will proceed in six related projects: a) three-dimensional structural determination of the crystalline enzyme; b) the role of monovalent cations in controlling substrate binding; c) the kinetics and mechanism of interaction during electron transfer between these proteins during monooxygenation; d) the factors which control oxygen activation and product formation; e) the metabolic activation of halohydrocarbons by cytochrome P-450; and f) the metabolic control of this complex enzyme system. Advantage will be taken of the knowledge gained with this bacterial enzyme system to gain a better understanding of the mammalian drug metabolism system. The techniques which will be used in this project include: a) recording spectrophotometry; b) stopped-flow spectrophotometry with kinetic analysis of the data obtained; c) electron paramagnetic resonance spectrometry; d) computer modeling of metabolic control; and e) radioisotope counting.